WO2011090159A1 - 蛋白質の分解活性を測定するためのプローブ試薬 - Google Patents
蛋白質の分解活性を測定するためのプローブ試薬 Download PDFInfo
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- the present invention relates to a fluorescent probe reagent for measuring a specific proteolytic activity depending on the ubiquitin-proteasome system in living cells.
- the ubiquitin-proteasome system is well known as one of the protein degradation pathways of cells.
- a plurality of small proteins called ubiquitin are added in a chain to a denatured protein or a protein with abnormal folding.
- This ubiquitin chain becomes a mark as a protein to be degraded, and is recognized and destroyed by the proteasome, which is a protein degradation apparatus.
- This system removes abnormal proteins in cells.
- the ubiquitin-proteasome system is not necessarily just a system for quality control of intracellular proteins. Even proteins that are structurally and functionally normal are decomposed according to the state of the cells at that time, and various cell functions are controlled by suppressing their activity.
- the light intensity is affected by factors that do not depend on the protein degradation activity, such as uneven distribution of the probe reagent and uneven expression, cell and tissue morphology, quenching due to fading, uneven excitation light, etc. Quantitative measurement is difficult.
- the amount of light decreases due to an increase in decomposition activity, there is a problem that the sensitivity of the detector is insufficient and the S / N is reduced, making accurate measurement difficult.
- CBG68 luciferase showing green luminescence derived from a beetle and expressed in cells.
- CBR luciferase showing red luminescence derived from the beetle was expressed in the same cells. The amount of luminescence of CBG68 increases or decreases according to the degradation activity of I ⁇ B ⁇ in the proteasome.
- CBR luminescence was not affected and used as a control. They measure luminescence in a group of cells cultured in multi-well plates. To correct differences in luminescence due to differences in the number of cells between wells, the degradation activity of I ⁇ B ⁇ is adjusted to green / red. It was measured as a ratio of emission intensity (Non-patent Document 4). Although this method has improved quantitativeness compared to single-wavelength photometry, in order to express two probe molecules individually, the ratio of their expression levels, that is, the ratio of the luminescence intensity, is made uniform between cells. There is a problem that it is difficult.
- luciferase and fluorescent protein used as labels there is a possibility that they are not uniformly distributed in the cells and the localization thereof is different. These are factors that reduce the accuracy, particularly when it is desired to measure the protein degradation activity at the single cell level.
- An object of the present invention is to provide a fluorescent probe reagent for measuring a specific proteolytic activity depending on the ubiquitin-proteasome system in living cells.
- Such a probe reagent overcomes the problems of conventional methods as described in the background art.
- the present invention includes the following features.
- Proteasome activity using the probe reagent according to any one of (1) to (6) above, the vector according to (8) above, or the transformed cell according to (9) or (10) above A method for screening a therapeutic agent for a disease associated with an abnormality in the ubiquitin-proteasome system, comprising measuring the activity of degrading the probe reagent protein in the ubiquitin-proteasome system in a cell in the presence of a candidate substance that controls the ubiquitin.
- the present invention provides a probe reagent capable of measuring in real time the protein degradation activity in the ubiquitin-proteasome system in living cells (also referred to as “live cells”).
- This probe reagent overcomes the problems caused by the probe reagents mentioned in the background art.
- a degradation-stopping peptide By disposing a degradation-stopping peptide between two fluorescent proteins, a signal from one fluorescent protein can be obtained even when the reagent is degraded due to an increase in the degradation activity of the protein. As a result, high-precision measurement can be performed even when the decomposition activity increases. Since the two fluorescent proteins are present in one molecule, they are expressed in a 1: 1 ratio in any cell.
- the structure of the probe reagent of the present invention is shown.
- the structure of a probe reagent for searching for a degradation-stopping peptide is shown.
- the site of p105 used as degradation stop peptide and spacer peptide is shown.
- An example of fluorescence observation of a probe reagent for searching for degradation-stopped peptides is shown.
- the result of the FRET measurement of the probe reagent for searching for a degradation stop peptide is shown.
- * indicates that fluorescence was observed not only in the nucleus but also in the cytoplasm.
- the time change of the fluorescence intensity ratio of the Geminin degron probe reagent in the cell nucleus and cytoplasm is shown.
- Figure 3 shows time-lapse imaging of a Geminin degron probe reagent. The structure of an I ⁇ B ⁇ degron probe reagent is shown. 2 shows time-lapse imaging of an I ⁇ B ⁇ degron probe reagent. The time change of the fluorescence intensity ratio of the I ⁇ B ⁇ degron probe reagent in cells No. 1 to No. 4 is shown.
- the probe reagent of the present invention is made of a protein, and as a structure, the fluorescent protein I from the N-terminal side, a peptide that stops the degradation of the protein, and a spacer peptide that provides a distance between the degradation-stopping peptide and the next fluorescent protein II , Fluorescent protein II, and a protein that undergoes degradation are linked to each other.
- this probe reagent undergoes degradation from the C-terminal side, but its degradation is stopped by the degradation-stopping peptide.
- the fluorescent protein I remains and the fluorescent protein II is decomposed and disappears. Therefore, the activity of degrading the protein can be measured by monitoring the change in the fluorescence intensity.
- the probe reagent of the present invention is characterized in that it can be used to measure proteolytic activity by a ubiquitin-proteasome system in living cells.
- the proteasome is a proteolytic device possessed by cells. It has a barrel-shaped structure and has an active site of protease in its lumen. Proteins that are degraded by the proteasome are modified by polyubiquitination by the action of ubiquitin ligase and the like. The proteasome recognizes the protein to which this mark is added, unwinds its three-dimensional structure, takes it into the lumen, and destroys it to a peptide of several amino acids.
- p105 which is one of the components of the transcription factor NF ⁇ B
- p50 is one of the components of the transcription factor NF ⁇ B
- p50 moves to the nucleus and promotes the transcriptional activity of the gene. That is, it can be said that NF ⁇ B uses the proteasome degradation activity to control ON / OFF of its transcriptional activity.
- the probe reagent of the present invention utilizes a limited degradation reaction in the proteasome, thus stopping the degradation of the protein in the middle. Assuming that the fluorescent protein having a ⁇ -barrel structure is tightly-folded-domain, and combining it with a peptide having a simple-sequence sequence, a probe reagent that stops the degradation in the proteasome was prepared.
- This probe reagent can be degraded by two types of fluorescent proteins with different excitation and / or fluorescence wavelengths, a peptide that stops degradation, a spacer peptide that keeps a distance between the degradation-stopping peptide and the next fluorescent protein, and proteasome It is composed of five regions of the protein to be received (referred to as “degron protein”).
- the probe reagent has a structure in which these five regions are linked as one amino acid chain. Each region is arranged in the order of fluorescent protein I, degradation-stopping peptide, spacer peptide, fluorescent protein II, and degron protein from the N-terminal side (FIG. 1). For example, when fluorescent proteins I and II of the probe reagent having different fluorescence wavelengths are used, this reagent emits two wavelengths of fluorescence when the degron protein is not degraded, because its full length is maintained. Can do. On the other hand, when the degron protein is decomposed, it is decomposed and disappears with the fluorescent protein II linked to the N-terminal side.
- the degradation activity of degron protein can be measured by measuring the change in the fluorescence intensity of the two wavelengths.
- Fluorescent proteins include Green fluorescent protein (GFP) obtained from Aequorea jellyfish, mutants thereof, and other biological species such as coral and mutants such as, but not limited to, GFP, EGFP, CFP, YFP , ECFP, YPet, CyPet, Venus, mCherry, Cerulean, mKeima, T-Sapphire, Midoriishi-Cyan, Kusabira-Orange, and other known fluorescent proteins can be used (Current Protocols .33 (John Willy & Sons), J. Endocriol. 2001; 170: 297-306, Bioorganic & Medicinal Chemistry Letters 2009; 19: 3748-3751). In order to measure the degradation activity of degron protein from changes in fluorescence intensity at two wavelengths, fluorescent proteins I and II are used which have greatly different excitation and / or fluorescence wavelengths, respectively.
- GFP Green fluorescent protein
- the fluorescent proteins I and II are arranged at such positions that the fluorescence energy transfer (FRET) occurs.
- FRET fluorescence energy transfer
- fluorescent protein II disappears due to degradation of degron protein
- FRET is eliminated. Therefore, by using a pair that can be a donor and acceptor in FRET as a fluorescent protein, protein degradation activity is also observed from changes in the amount of FRET. Can be measured.
- the amount of FRET changes, the fluorescence intensity of each donor and acceptor when the donor is excited is changed.
- a change in FRET amount can be measured as a change in the ratio of the fluorescence intensities of fluorescent proteins I and II when fluorescent protein I is excited.
- FRET fluorescence lifetime
- YFP Yellow fluorescent protein
- the peptide for stopping the degradation of the probe reagent was searched for from the region that seems to stop the degradation of p105, a protein that undergoes limited degradation in the proteasome (the amino acid number of human p105 is Accession below) NM_001165412 (human NFKB1 transcript, variant 2), NM_008689 and NM_001159394 (all of which are mouse-homologs) are also known).
- the human p105 protein is composed of 969 amino acids. As mentioned earlier, when this protein is activated, about half of the C-terminal region of the proteasome is degraded, but up to 435 amino acids from the N-terminus are prevented from degradation and a protein called p50 is obtained. Released.
- GRR Glycine Rich Region
- Simple sequence is a region of amino acids 376-404 from the N-terminal side, and 19 glycines are concentrated in this (Orian, A. et al., Mol Cell. Biol., Vol. 19, 366p. 3664-3673, 1999). Therefore, it was examined whether this GRR and its surrounding sequence function as a degradation-stopping peptide even before the fluorescent protein. GRR alone does not stop the degradation, and the termination of degradation further includes the C-terminal sequence. Was found to be necessary. It was also found that the amino acid sequence following the C-terminal side of GRR does not necessarily have to be derived from p105.
- Such a peptide having the ability to stop degradation may be another protein that undergoes limited degradation in the proteasome, such as p105.
- proteins include, but are not limited to, for example, p100 (transcription factor, a component of NF ⁇ B), Cubitus interrupttus (Drosophila transcription factor; NM_079878, NM_001081125 (mouse homolog), NM_005270 (human homolog)), EBNA -1 (Epstein-Barr virus protein), Spt23 (yeast transcription factor; for example NC_001143, NM_001179586, _NC_006029, EU861367), Mga2 (yeast transcription factor; for example NM_001179555, NC_001141, NC_006029, CP000499), etc.
- GRR-like peptides derived from these can also be used as degradation-stopping peptides.
- Such degradation-stopping peptides can have the following characteristics (a) to (d).
- Degradation-stopping peptide consists of a peptide containing 70% or more of an amino acid having 0 to 3 carbon atoms in the side chain, such as glycine, alanine, serine, aspartic acid, and asparagine.
- a plurality of the degradation-degrading peptides may be arranged between the fluorescent protein I and the spacer.
- spacer peptide is a peptide for keeping a distance between the degradation-stopping peptide and the fluorescent protein II and includes one or more amino acids, for example, 1 to 200 amino acids, preferably 2 to 100 amino acids, more preferably Consists of 5 to 50 amino acids.
- the number of amino acids between the fluorescent protein I and the degradation-stopping peptide is less than 10 and may be 0.
- Degron protein is a protein that is degraded by the ubiquitin-proteasome system to be measured. Many proteins that are degraded by the proteasome are known, and the probe reagent of the present invention can be used for measuring the degradation activity of these proteins by replacing this region.
- proteins that are degraded in the ubiquitin-proteasome system include, but are not limited to, Cyclin (A, B, D, E), p53, A ⁇ , p27, p21, p16, p15, p18, p19, p62, I ⁇ B, NF- ⁇ , c-fos / c-jun, c-myc, ⁇ catenin, E2F-1, p130, cdc25, Tyrosine amino transferase, Polo-like kinase, Topoisomerase 1, Smad, Notch, Nrf2, HIF-1 ⁇ , Geminin, etc. (Adams, J. et al., Invest.
- the full-length structure of the degron protein may be used, but the degradation of the protein, such as the part that undergoes ubiquitination in the molecule and the part that undergoes modifications such as phosphorylation necessary to induce the ubiquitination You may use only the part which is indispensable. Probe reagents become foreign molecules for cells. Therefore, in order to avoid unnecessary disturbance in the cell activity due to the introduction of the probe reagent into the cell, it is preferable that the degron protein contains only the structure necessary for degradation and other active regions are omitted.
- abnormalities in the ubiquitin-proteasome system are associated with various diseases.
- diseases for example, neurodegenerative diseases, cancer (or tumors), ischemic diseases (for example, infarcts), inflammatory diseases, allergic diseases and the like are known.
- diseases associated with such abnormalities for example, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease occur because degron proteins are difficult to be degraded by the proteasome (JP 2009-149524, JP 2008-222603, etc.).
- An agent that activates the proteasome can be a therapeutic agent, while cancer is caused by degradation of the degron protein by the proteasome (Japanese Patent Application Laid-Open No. 2007-254320, etc.), and thus a proteasome inhibitor can be a therapeutic agent.
- the probe reagent of the present invention can be used to measure the degradation reaction of a protein by a ubiquitin-proteasome system possessed by a cell. Since this degradation system is universally present in eukaryotic cells, any probe reagent can be used. Various types of cells can be measured. Such cells also include cells associated with abnormalities in the ubiquitin-proteasome system, such as nerve cells, tumor cells, lymphocytes, skin cells, and synovial cells.
- the probe reagent of the present invention is composed only of a protein
- a nucleic acid for example, gene, DNA or messenger RNA
- the probe reagent is expressed in the cell for measurement.
- Proteasomes are known to be distributed in the nucleus and cytoplasm, but signal sequences that are localized and expressed only in the nucleus (nuclear localization signal) or signal sequences that are localized and expressed only in the cytoplasm (extranuclear)
- the export signal By adding the export signal) to the probe reagent and expressing it, the degradation activity of the degron protein at these sites can be selectively measured.
- the nuclear localization signal and the nuclear export signal those known in the literature can be used.
- For introduction of DNA or RNA into cells commonly used techniques such as lipofection, electroporation, and microinjection can be used.
- the nucleic acid encoding the probe reagent of the present invention is obtained by, for example, obtaining a DNA encoding each of the proteins and peptides constituting the probe reagent by a known cloning method, PCR method, etc. Can be produced. In cloning, the nucleic acid can be inserted into an appropriate vector so that it can be expressed and cloned into cells such as Escherichia coli, fungi, plant cells, and animal cells. Vectors are, for example, plasmids, phages, cosmids, viruses and the like.
- nucleic acid an expression cassette linked with regulatory sequences such as promoter, enhancer, replication origin, ribosome binding site, terminator, polyadenylation site can be formed and inserted into the multicloning site of the vector.
- regulatory sequences such as promoter, enhancer, replication origin, ribosome binding site, terminator, polyadenylation site.
- Techniques such as gene recombination technology, transformation method, transfection method, and PCR method are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second. 1989 (Ausubel et al., Short Protocols in Molecular Biology, John John Wiley & Sons (2002), etc., and can be used for the present invention.
- cells that retain the nucleic acid and stably express the probe reagent are prepared and used for measurement. Also good. Furthermore, by producing a transgenic non-human organism (for example, a non-human animal) into which this nucleic acid has been introduced, protein degradation activity can be measured at the individual level. At that time, by connecting the nucleic acid downstream of an appropriate promoter, it can be selectively expressed in the target organ or tissue of the non-human animal and used for the measurement.
- the non-human animal can be produced according to a known technique, for example, after introducing the nucleic acid into the genome of an animal-derived embryonic stem (ES) cell or induced pluripotent stem (iPS) cell so that the nucleic acid can be expressed. This is carried out by transplanting an embryo obtained by introducing iPS cells into a blastocyst into a foster mother's uterus and giving birth to obtain a chimeric non-human animal and further offspring animals.
- ES animal-derived embryonic stem
- iPS induced pluripotent stem
- fluorescent proteins examples include Cerulean (excitation peak 433 nm, fluorescence peak 475 nm) and mKeima (excitation peak 440 nm, fluorescence peak 620 nm). In the case of measurement using FRET, this one-wavelength excitation-two-wavelength fluorescence photometry is used. If both the excitation and fluorescence wavelengths are greatly different, two-wavelength excitation-two-wavelength fluorescence photometry is performed in which measurement is performed by switching both of them. Examples of such fluorescent proteins include Venus (excitation peak 515 nm, fluorescence peak 528 nm) and mCherry (excitation peak 587 nm, fluorescence peak 610 nm).
- the ratio method cancels changes in fluorescence intensity that do not depend on protein degradation, such as differences in the distribution of probe reagents in cells, uneven illumination of excitation light, and fading of fluorescence. Measurement is possible.
- a microscope imaging system in which a detector such as a cooled CCD camera is connected to a fluorescence microscope can be used.
- a filter switching device When switching the excitation wavelength according to the measurement method, a filter switching device, a monochromator, or the like is connected after the light source.
- a filter switching device and a two-wavelength spectrometer for imaging are connected in front of the detector.
- a dual-band filter that matches the wavelength characteristics of the two fluorescent proteins used for the filter and dichroic mirror of the fluorescence microscope, and combine this with a color camera. Good.
- the degradation activity of the degron protein is imaged as a color change, the detection becomes easy.
- a microscope imaging device a laser scanning confocal microscope, a multiphoton excitation microscope, or the like can be used. Single-cell resolution is not required, and if you want to obtain data from many cells, you can use a fluorescence spectrophotometer, plate reader, flow cytometry, etc. In order to perform measurement at the individual level, a macro imaging device using a dark box can be used.
- the reagent can be used in the medical field as follows, for example.
- the probe reagent of the present invention can be used universally for these proteins by replacing the degron protein portion.
- the probe reagent of the present invention contributes to the development of therapeutic methods and therapeutic agents for these diseases by measuring the degradation activity in cells or individual animals by using the degron protein as the cause or disease-related protein of the disease. Expected to do.
- the present invention further uses the probe reagent, the vector, or the transformed cell to detect the probe reagent protein in the ubiquitin-proteasome system in a cell in the presence of a candidate substance that controls proteasome activity.
- a method for screening for a therapeutic agent for a disease associated with an abnormality of the ubiquitin-proteasome system comprising measuring a degradation activity.
- Diseases associated with abnormalities in the ubiquitin-proteasome system include neurodegenerative diseases such as those exemplified above, cancer (or tumor), ischemic diseases (for example, infarcts), inflammatory diseases, allergic diseases, etc. Abnormalities are associated with abnormal regulation of proteasome activity.
- proteasome activators can be therapeutic agents in neurodegenerative diseases
- proteasome inhibitors can be therapeutic agents in cancer and ischemic diseases (such as infarcts).
- Uru (Special Table 2002-541206, Special Table 2001-511814, Special Table 2008-525427, etc.).
- the probe reagent or vector of the present invention is brought into contact with a candidate substance by contacting a cell or transformed cell, particularly a cell associated with an abnormality of the ubiquitin-proteasome system, with the ubiquitin-proteasome system contained in the cell.
- a substance that controls (i.e., enhances or suppresses (inhibits)) the degradation activity of the probe protein is selected.
- the degradation activity of the probe reagent protein can be measured, for example, as a change in the fluorescence intensity ratio between the fluorescent protein I and the fluorescent protein II.
- the present invention further includes an abnormality of a ubiquitin-proteasome system and a disease, which comprises contacting the probe reagent or vector with a cell or cell extract from a diseased patient and measuring the degradation activity of the probe reagent protein.
- an abnormality of a ubiquitin-proteasome system and a disease which comprises contacting the probe reagent or vector with a cell or cell extract from a diseased patient and measuring the degradation activity of the probe reagent protein.
- a normal cell or a cell extract of the cell is used as a control.
- abnormalities in the ubiquitin-proteasome system can be correlated with diseases.
- a disease can be selected from the diseases exemplified above.
- the degron protein of the probe reagent is a protein associated with a disease, such as Cyclin (A, B, D, E), p53, A ⁇ , p27, p21, p16, p15, p18, p19, p62, I ⁇ B, NF- ⁇ , c-fos / c-jun, c-myc, ⁇ catenin, E2F-1, p130, cdc25, Tyrosine amino transferase, Polo-like kinase, Topoisomerase 1, Smad, Notch, Nrf2, HIF-1 ⁇ , Geminin, etc. Is a protein.
- a disease such as Cyclin (A, B, D, E), p53, A ⁇ , p27, p21, p16, p15, p18, p19, p62, I ⁇ B, NF- ⁇ , c-fos / c-jun, c-myc, ⁇ cat
- Example 1 ⁇ Search for peptides to stop degradation>
- a peptide sequence having an ability to stop degradation from p105 was searched.
- a probe reagent using CyPet as fluorescent protein I, YPet as fluorescent protein II, and Geminin as a degron protein was prepared (FIG. 2).
- CyPet is a fluorescent protein derived from CFP, with an excitation wavelength peak of 435 nm and a fluorescence wavelength peak of 477 nm.
- YPet is a fluorescent protein derived from YFP, with an excitation wavelength peak of 517 nm and a fluorescence wavelength peak of 530 nm.
- Geminin is one of the factors that regulate cell cycle progression and is a protein that has the function of inhibiting the licensing of DNA replication (Cell 1998; 93 (11): 1043-1053, Am. J. Pathol. 2002; 161 (1): 267-273). Its abundance is strictly controlled during the cell cycle, and the expression level increases in the S / G 2 / M phase, and disappears in the G 1 phase by promoting degradation by the ubiquitin-proteasome system.
- Geminin structure (for example, NM_015895 (human, SEQ ID NO: 18 and 19), NM_020567 (mouse)) consists of amino acids 1-110 on the N-terminal side containing the necessary portion for degradation of this molecule. Part was used (Geminin (1/110)).
- the cDNA of each of the above molecules was amplified by PCR, and ligated by inserting into each restriction enzyme site of the cloning vector, multi-cloning site of pBluescript®II SK (+) (Stratagene) to prepare a gene DNA of a probe reagent.
- the sequence of the peptide for stopping the degradation was searched from the region of amino acids 366-440 of human-derived p105 (SEQ ID NOs: 16 and 17). This area includes GRR and processing points.
- FIG. 3 shows a schematic diagram thereof (p105 (m / n) indicates a peptide consisting of amino acids m to n from the N-terminus of p105). Furthermore, the gene DNA of the prepared probe reagent was excised from the pBlueScript vector using a restriction enzyme, and this was inserted into the expression vector, CSII-EF-MCS multicloning site to prepare a vector for expression in cultured cells. .
- HeLa cells were cultured in a 35 mm ⁇ glass bottom dish in Dulbecco's modified Eagle medium containing 10% fetal bovine serum.
- the prepared vector plasmid was mixed with a transfection reagent, FuGENE (Roche Applied Science), and added to the medium.
- HeLa cells were cultured for 2 days in a CO 2 incubator to express the probe reagent and to experience the cell cycle.
- Geminin is a protein that functions in the nucleus and has a nuclear localization signal in the molecule, so that the probe reagent is selectively incorporated into the nucleus. Geminin changes its amount periodically according to the cell cycle by promoting and suppressing degradation by the ubiquitin-proteasome system.
- an inverted microscope (IX70, Olympus) was used, and fluorescence images were acquired with a three-plate cooled CCD color camera (ORCA3CCD, Hamamatsu Photonics). With this camera, images of the three wavelength regions of red, green, and blue can be acquired simultaneously and displayed together to observe a color image. Moreover, the brightness
- CyPet fluorescence is mainly blue and partly green due to its spectral characteristics. Since most of the fluorescence of YPet falls in the green region, FRET measurement is performed by calculating the ratio of both the luminance of the image in the blue wavelength region as CyPet and the luminance of the image in the green wavelength region as the fluorescence intensity of YPet. Went. Moreover, it excited with the bandpass filter of 490 nm, and acquired fluorescence with the bandpass filter of 510-560 nm. In this setting, since the fluorescence when YPet was directly excited could be observed in the green wavelength region, the presence and localization of YPet were confirmed. The luminance analysis of the image was performed with the image analysis device AQUACOSMOS (Hamamatsu Photonics).
- FIG. 4 shows an observation example of cells expressing a probe reagent using p105 (376/440) and p105 (405/440) as peptides for stopping the degradation.
- the fluorescence was distributed only in the nucleus in all the cells observed.
- only weak fluorescence was observed in the blue wavelength region when CyPet was excited, whereas strong fluorescence was observed in the green wavelength region, indicating that strong FRET from CyPet to YPet occurred.
- the ratio of the fluorescence intensity of YPet / CyPet when CyPet was excited was 7.64 ⁇ 1.03 (mean ⁇ standard deviation).
- amino acid chain does not have to be derived from p105.
- 30 a.a. is shown in SEQ ID NO: 15. From these results, it was found that a peptide sequence as a spacer that separates GRR from its C-terminal side and fluorescent protein II is necessary to stop the decomposition of the probe reagent. In addition, from the result of p105 (366/440) between the N-terminal side of GRR and fluorescent protein I, it was found that the degradation was not stopped when 10 amino acids were inserted.
- the following shows the amino acid sequence of the degradation-stopping peptide + spacer peptide having the ability to stop the degradation of the probe reagent extracted in this experiment, and the base sequence of DNA.
- p105 (376/440) 376-440 amino acid sequence of human p105 (SEQ ID NO: 1) Decomposition stop peptide: 376-404th (underlined), spacer peptide: 405-440th GGGSGAGAGGGGMFGSGGGGGGTGSTGPG YSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTES Base sequence (SEQ ID NO: 2) GGCGGTGGTAGTGGTGCCGGAGCTGGAGGCGGAGGCATGTTTGGTAGTGGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGG TATAGCTTCCCACACTATGGATTTCCTACTTATGGTGGGATTACTTTCCATCCTGGAACTACTAAATCTAATGCTGGGATGAAGCATGGAACCATGGACACTGAATCT p105 (382/440) 382-440 amino acid sequence of human p105 (SEQ ID NO: 3) Decomposition stop peptide: 382-404th (underlined), spacer peptide: 405-440th GAGGGGMFG
- Fluorescent protein I is released into the cytoplasm as the cell cycle progresses. Protein II was also observed using Venus and mCherry, AmCyan and mCherry, TurboGFP and TurboRFP, mAzami-Green and mKusabira-Orange, respectively. From this result, it is considered that many types of fluorescent proteins can be applied as constituents of the probe reagent.
- Example 2 ⁇ Time-lapse imaging with probe reagent using Geminin (1/110) as degron protein> Peptide p105 (376/440) (decomposition-stopping peptide p105 (376/404) + spacer peptide p105 (405/440)) with the ability to stop degradation, and CyPet and YPet as fluorescent proteins I and II as degron proteins Time-lapse imaging was performed on HeLa cells in which a probe reagent using Geminin (1/110) was expressed, and the temporal change in fluorescence with the cell cycle was measured.
- Measurement was performed with an incubator fluorescence microscope (LCV110, Olympus). Upon excitation with a 455 nm LED, CyPet fluorescence images were acquired with a cooled CCD camera through a 460-510 nm bandpass filter and YPet fluorescence images with a 515-560 nm bandpass filter. Images from direct excitation of YPet were acquired with a 528-555 nm bandpass filter by excitation with a 505 nm LED. Each image was acquired every 30 minutes for 48 hours. Luminance analysis was performed separately for the nuclear region and the other cytoplasmic region, and the ratio of the fluorescence intensity of YPet / CyPet when CyPet was excited was measured.
- Figure 6 shows the results.
- the value of the fluorescence intensity ratio decreased rapidly with cell division in both the nucleus and cytoplasm.
- the ratio value gradually recovered in the nucleus, but the decreased value was maintained in the cytoplasm.
- CyPet fluorescence was always observed in the nucleus or cytoplasm, but YPet fluorescence rapidly decreased with cell division, and then It was observed that the nucleus slowly recovered (Fig. 7).
- I ⁇ B ⁇ is a protein that binds to NF ⁇ B in the cytoplasm and inhibits its nuclear translocation and transcriptional activity.
- I ⁇ B kinase is activated, which phosphorylates I ⁇ B ⁇ .
- Phosphorylated I ⁇ B ⁇ undergoes ubiquitination by ubiquitin ligase and is degraded by the proteasome.
- CyPet was used for fluorescent protein I
- YPet was used for fluorescent protein II
- full length I ⁇ B ⁇ was used as a degron protein
- p105 (376/404) was used as a degradation-stopping peptide
- p105 (405/440) was used as a spacer peptide.
- Probe by replacing Geminin (1/110), the degron protein of the probe reagent for searching for the above-mentioned degradation-stopping peptides, with human or mouse-derived I ⁇ B ⁇ (human: NM_020529, NM_003340 or NM_003339, mouse: AF112979)
- a reagent was prepared (FIG. 8).
- Cos7 cells were cultured in a 35 mm ⁇ glass bottom dish in Dulbecco's modified Eagle medium containing 10% fetal bovine serum. Similar to the experiment for searching for the degradation-stopping peptide, the gene DNA of the probe reagent was transfected and imaged with an incubator microscope. Images were acquired every 10 minutes for 2 hours. Ten minutes after the start of imaging, TNF- ⁇ , an agent that activates NF ⁇ B, was added to the medium to a concentration of 20 ng / ml.
- FIG. 9 shows the time change of the CyPet fluorescence image with CyPet excitation and the YPet fluorescence image with YPet excitation.
- One cell (No. 1) out of four cells entering the field showed a response, and a decrease in fluorescence intensity was observed only with YPet.
- a decrease in the fluorescence intensity ratio of YPet / CyPet when CyPet was excited was also observed (FIG. 10). This result indicates that the degradation process of I ⁇ B ⁇ by TNF- ⁇ stimulation can be visualized as a change in fluorescence with this probe reagent.
- the probe reagent of the present invention can contribute to the development of therapeutic methods and drugs for these diseases by measuring the degradation activity of proteins related to diseases caused by abnormalities in the ubiquitin-proteasome system in cells or individual animals. it can.
- SEQ ID NO: 13 Synthetic peptide
- SEQ ID NO: 14 DNA encoding the synthetic peptide
- SEQ ID NO: 15 CyPet-derived peptide
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Abstract
Description
<分解停止させるペプチドの探索>
目的のプローブ試薬の開発のために、p105から分解停止させる能力をもつペプチド配列を探索した。実験用のプローブ試薬として、蛍光蛋白質IにCyPet,蛍光蛋白質IIにYPet,デグロン蛋白質としてGeminin を使用したプローブ試薬を作製した(図2)。CyPetはCFPから派生した蛍光蛋白質で、励起波長ピークは435nm、蛍光波長ピークは477nmである。YPetはYFPから派生した蛍光蛋白質で、励起波長ピークは517nm、蛍光波長ピークは530nmである。このペアはCyPetをドナー、YPetをアクセプタとして効率よくFRETを起こすことが知られている。Gemininは細胞周期の進行を制御する因子の1つで、DNA複製のライセンス化を阻害する機能をもつ蛋白質である(Cell 1998; 93(11):1043-1053, Am. J. Pathol. 2002; 161(1):267-273)。その存在量は細胞周期の間で厳密に制御されており、S/G2/M期に発現量が増加し、G1期にはユビキチン-プロテアソーム系による分解が促進されることで消失する。この実験ではGemininの構造(例えばNM_015895 (human, 配列番号18及び19), NM_020567 (mouse))のうち、この分子の分解に必要な部分が含まれるN末端側の1-110番目のアミノ酸からなる部分を使用した(Geminin (1/110))。
ヒトp105の376-440番目のアミノ酸配列(配列番号1)
分解停止ペプチド:376-404番目(下線部),スペーサーペプチド:405-440番目
GGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTES
塩基配列(配列番号2)
GGCGGTGGTAGTGGTGCCGGAGCTGGAGGCGGAGGCATGTTTGGTAGTGGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGGTATAGCTTCCCACACTATGGATTTCCTACTTATGGTGGGATTACTTTCCATCCTGGAACTACTAAATCTAATGCTGGGATGAAGCATGGAACCATGGACACTGAATCT
p105 (382/440)
ヒトp105の382-440番目のアミノ酸配列(配列番号3)
分解停止ペプチド:382-404番目(下線部),スペーサーペプチド:405-440番目
GAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTES
塩基配列(配列番号4)
GGAGCTGGAGGCGGAGGCATGTTTGGTAGTGGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGGTATAGCTTCCCACACTATGGATTTCCTACTTATGGTGGGATTACTTTCCATCCTGGAACTACTAAATCTAATGCTGGGATGAAGCATGGAACCATGGACACTGAATCT
p105 (392/440)
ヒトp105の392-440番目のアミノ酸配列(配列番号5)
分解停止ペプチド:392-404番目(下線部),スペーサーペプチド:405-440番目
GGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTES
塩基配列(配列番号6)
GGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGGTATAGCTTCCCACACTATGGATTTCCTACTTATGGTGGGATTACTTTCCATCCTGGAACTACTAAATCTAATGCTGGGATGAAGCATGGAACCATGGACACTGAATCT
p105 (376/434)
ヒトp105の376-434番目のアミノ酸配列(配列番号7)
分解停止ペプチド:376-404番目(下線部),スペーサーペプチド:405-434番目
GGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHG
塩基配列(配列番号8)
GGCGGTGGTAGTGGTGCCGGAGCTGGAGGCGGAGGCATGTTTGGTAGTGGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGGTATAGCTTCCCACACTATGGATTTCCTACTTATGGTGGGATTACTTTCCATCCTGGAACTACTAAATCTAATGCTGGGATGAAGCATGGA
p105 (376/420)
ヒトp105の376-420番目のアミノ酸配列(配列番号9)
分解停止ペプチド:376-404番目(下線部),スペーサーペプチド:405-420番目
GGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITF
塩基配列(配列番号10)
GGCGGTGGTAGTGGTGCCGGAGCTGGAGGCGGAGGCATGTTTGGTAGTGGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGGTATAGCTTCCCACACTATGGATTTCCTACTTATGGTGGGATTACTTTC
p105 (376/409)
ヒトp105の376-409番目のアミノ酸配列(配列番号11)
分解停止ペプチド:376-404番目(下線部),スペーサーペプチド:405-409番目
GGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPH
塩基配列(配列番号12)
GGCGGTGGTAGTGGTGCCGGAGCTGGAGGCGGAGGCATGTTTGGTAGTGGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGGTATAGCTTCCCACAC
p105 (376/404)+30 a.a. (配列番号13)
ヒトp105の376-404番目のアミノ酸配列に、CyPetのC末端付近の30個のアミノ酸を加えた配列
分解停止ペプチド:p105の376-404番目(下線部),スペーサーペプチド:グルタミン酸+フェニルアラニン+CyPetの211-238番目のアミノ酸
GGGSGAGAGGGGMFGSGGGGGGTGSTGPGEFDPNEKRDHMVLLEFVTAAGITLGMDELY
塩基配列(配列番号14)
GGCGGTGGTAGTGGTGCCGGAGCTGGAGGCGGAGGCATGTTTGGTAGTGGCGGTGGAGGAGGGGGCACTGGAAGTACAGGTCCAGGGGAATTCGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTAC
Geminin (1/110)をデグロン蛋白質、分裂停止ペプチド+スペーサーペプチドをp105 (376/440)としたプローブ試薬で、細胞周期の進行にともなう蛍光蛋白質Iの細胞質への放出は、蛍光蛋白質Iと蛍光蛋白質IIにそれぞれVenusとmCherry,AmCyanとmCherry,TurboGFPとTurboRFP,mAzami-GreenとmKusabira-Orangeを使用したものでも観察された。この結果から、このプローブ試薬の構成要素として多くの種類の蛍光蛋白質が適用できると考えられる。
<Geminin (1/110)をデグロン蛋白質としたプローブ試薬でのタイムラプスイメージング>
分解停止能が示されたペプチドp105 (376/440) (分解停止ペプチドp105(376/404)+スペーサーペプチドp105(405/440))、および蛍光蛋白質I,IIとしてCyPet,YPetを、デグロン蛋白質としてGeminin (1/110)を使用したプローブ試薬を発現させたHeLa細胞でタイムラプスイメージングを行い、細胞周期にともなう蛍光の時間変化を測定した。
<IκBαをデグロン蛋白質としたプローブ試薬>
デグロン蛋白質をIκBαとして、その分解活性から転写因子NFκBの活性をモニタするためのプローブ試薬を作製した。IκBαは細胞質でNFκBと結合し、その核移行および転写活性を阻害している蛋白質である。細胞がNFκBを活性化させるシグナルを受けると、IκBキナーゼが活性化され、これがIκBαをリン酸化する。リン酸化を受けたIκBαはユビキチンリガーゼによりユビキチン化を受け、プロテアソームで分解される。これによりNFκBに対する抑制が解除され、核への移行、遺伝子の転写が活性化される。これまでに悪性腫瘍細胞や自己免疫疾患において、恒常的なNF-κBの活性化が報告されている。したがって、このような測定系はこれらの疾患の診断や治療薬の開発に応用できると考えられる。
配列番号14:合成ペプチドをコードするDNA
配列番号15:CyPet由来のペプチド
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
Claims (15)
- N末側からC末端側に向かって順番に、蛍光蛋白質I、蛋白質の分解を停止させるペプチド(分解停止ペプチド)、スペーサーペプチド、蛍光蛋白質II、および分解を受ける蛋白質のそれぞれのアミノ酸配列を含んでなるプローブ試薬であって、ここで該分解を受ける蛋白質がユビキチン-プロテアソーム系で分解される蛋白質であり、および、該プローブ試薬のC末端側から分解を受けるがその分解は分解停止ペプチドで停止することを特徴とするプローブ試薬。
- 蛍光蛋白質Iおよび蛍光蛋白質IIは、励起波長、蛍光波長、または、励起波長と蛍光波長の両方が異なるものである、請求項1に記載のプローブ試薬。
- 蛍光蛋白質Iおよび蛍光蛋白質IIはそれぞれ、蛍光エネルギー移動(FRET)でのドナーとアクセプタである、請求項1または2に記載のプローブ試薬。
- 核局在化シグナルまたは核外搬出シグナルをさらに含む、請求項1~3のいずれか1項に記載のプローブ試薬。
- 分解停止ぺプチドが、蛍光蛋白質Iとスペーサーの間に1個もしくは複数個存在する、請求項1~4のいずれか1項に記載のプローブ試薬。
- スペーサーペプチドは、分解停止ペプチドと蛍光蛋白質IIの間に距離をおくための1個以上のアミノ酸からなるペプチドである、請求項1~5のいずれか1項に記載のプローブ試薬。
- 請求項1~6のいずれか1項に記載のプローブ試薬をコードする核酸。
- 請求項7に記載の核酸を発現可能に含むベクター。
- 請求項8に記載のベクターを含む形質転換細胞。
- 疾病細胞である、請求項9に記載の形質転換細胞。
- 請求項1~6のいずれか1項に記載のプローブ試薬、請求項8に記載のベクター、あるいは請求項9または10に記載の形質転換細胞を用いて、プロテアソーム活性を制御する候補物質の存在下で細胞におけるユビキチン-プロテアソーム系での該プローブ試薬蛋白質の分解活性を測定することを含む、ユビキチン-プロテアソーム系の異常に関連する疾病の治療剤をスクリーニングする方法。
- プローブ試薬蛋白質の分解活性を、蛍光蛋白質IとIIの蛍光強度の比の変化として測定する、請求項11に記載の方法。
- 細胞がユビキチン-プロテアソーム系の異常に関連する疾病細胞である、請求項11または12に記載の方法。
- 請求項1~6のいずれか1項に記載のプローブ試薬または請求項8に記載のベクターと、疾病患者からの細胞または細胞抽出液とを接触させて該プローブ試薬蛋白質の分解活性を測定することを含む、ユビキチン-プロテアソーム系の異常と疾病との関連を調べる方法。
- プローブ試薬内の分解を受ける蛋白質(デグロン蛋白質)が疾病に関連する蛋白質である、請求項11または14に記載の方法。
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WO2021054464A1 (ja) | 2019-09-18 | 2021-03-25 | ダイキン工業株式会社 | 冷凍装置の室外機 |
WO2021066157A1 (ja) | 2019-10-04 | 2021-04-08 | 国立研究開発法人理化学研究所 | 新規蛍光タンパク質、及びその利用 |
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